Gangliosides with a modified acyl function

ABSTRACT

The invention relates to a ganglioside mixture, consisting of gangliosides, of the following general formula: (sugar)-OCH 2 —CH(—NH—CO—R 1 )—CH(OH)—CH═CH—R 2 , wherein (sugar) represents a sugar radical, the group —CO—R 1  represents an acyl-fatty acid which is bonded to the radical of the molecule in the form of an amide, R 1  represents a straight saturated alkyl radical having at least 10 C-atoms and R 2  represents a straight, saturated alkyl radical having at least 10 C-atoms or a straight alkenyl radical having at least 10 C-atoms and one, two or three double bond(s). Said mixture is characterised in that at least 10 wt. % of the gangliosides of the general formula I are of the group —CO—R 1  and the acyl-fatty acids are a C20:0 fatty acid. Said mixtures exhibit an improved biological activity.

The invention relates to a ganglioside mixture, the use of thisganglioside mixture for preparing a dietetic, pharmaceutical and foodcomposition, as well as a corresponding composition.

Gangliosides are glycolipids of a complicated structure, which arepredominantly located on the outer surface of the plasma membrane of acell. They contain a polar part (head) made up of sugars and sugarderivatives and, in some cases, N-acetyl neuraminic acid, as well asseveral nonpolar aliphatic chains made up of fatty acids and theirderivatives.

The structure of the ganglioside oligosaccharide chain directlydetermines the cell-cell interaction (cell adhesions), the receptorcoverage (e.g. IL-2, selectines), cf. Robb, R. J., J. Immunol. 1986,136, 971-976, enterotoxin linkage (antibacterial/antiviral and prebioticeffects), cf. Rueda, R., Sabetel, J. L., Maldonaldo, J., Molinos-Font,J. A., Gil, A. J. Pediatr. 1998, 133, 90-94, adhesion/antibody linkage,cf. Welte, K., Miller, G., Chapman, P. B., Yuasa H., Natoli, E.,Kunicka, J. E., Cordon-Cardo, C., Buhrer, C., Old, L. J., Houghton, A.N., J. Immunol. 1987, 139, 1763-1771 and cellular signal transduction(GM3 in the case of T-cells), cf. Rippo, M. R., Malisan, F., Ravagnan,L., Tomassini, B., Condo, I., Constantini, P., Susin, S. A., Rufini, A.,Todaro, M., Kroemer, G., Testi, R. FASEB J. 2000, 14, 2047-2057.

The fatty acid composition is however responsible for the spatialarrangement of the molecules within a membrane (micro-domainformation/clustering), and is a co-determinant for thesubstrate-specific reaction of glycolipids in the metabolism(enzyme-substrate specificity).

When the literature refers to fatty acid compositions in the context ofgangliosides, very often no distinction is made between the two nonpolaraliphatic chains, both of which are called fatty acids. These fattyacids, however, are a structure-determining and function-determiningelement, cf. Ladisch, S., Hasegawa, A.; Li, R., Kiso, M. Biochemistry1999, 34, 1197-1202. The N-acyl sphingosine portion of gangliosides(ceramide) is of importance as an intracellular signal-mediatingmolecule. The long-chain acyl groups of gangliosides are omnipresentfatty acids in biological systems, and impart these gangliosides a highbio-activity in their manifold functions.

Gangliosides are enzymatically (lyosomal enzymes) intracellularlyconverted into ceramide, this is converted into sphingomyelin and thento sphingosine, sphingosine-1P. Ceramide, via sphingomyelin conversion,also influences the synthesis of diacyl glycerol and phosphoryl diacylglycerol. These substances all are highly potent signal-mediatingmolecules. Gangliosides and gangliosides converted into ceramide thusdetermine the metabolism and the cell function in manifold ways. Whilethe 3-keto bound fatty acid of the ganglioside structurally andfunctionally co-determines all metabolites of the ceramide, e.g.sphingosine, i.e. this aliphatic chain remains unchanged in themetabolism, the N-acylated fatty (amid) acid is only contained as afatty acid composition of the ceramide itself and corresponding glycosylderivatives, and is cleaved off during the metabolism of secondarymessengers.

The specific acylation of the molecule determines the potency ofgangliosides/ceramides and their metabolites as signal-mediatingmolecules. The metabolism of ganglioside derivatives bysubstrate-specific enzymes (sphingomyelinase, ceramidase, sphingosinacyltransferase) and the enzymatic regulation of the ceramide activity as asignal molecule (ceramide hydrogenase, ceramide kinase) is co-determinedby the acyl pattern as a structural element. In addition, saturatedfatty acids having a carbon length of up to C24 cause rapid gangliosidemetabolism (de novo synthesis/conversion). The specific acylation of themolecule is also relevant for the influence of gangliosides on variousintracellular regulatory enzymes (phosphokinase C, phospholipase D andvarious protein phosphatases/kinases).

Hitherto known biological functions of gangliosides are:

-   -   Development of the gastrointestinal tract of newborn babies;        development of the intestinal immunosystem, cf. Rueda, R.,        Maldonado, J., Narbona, E., Gil, A. Early human Development        1998, 53, p 135-147; Jensen, R. Lipids 1999, 34, 1243-1271.    -   Anti-carcinogenic effects (induces apoptosis, stops cell        growth), cf. Vesper, H., Schmelz, E.-M.,        Nikolova-Karakashian, M. N., Dillehay, D. L., Lynch, D. V.,        Merril, A. H. Jr. J. Nutr. 1999, 129, 1239-1250; Farooqui, A.,        Harrocks, L. A., Farooqui, T., Chemistry and Physics 2000, 106,        1-29.    -   T-cell differentiation is induced (induction of the cell        maturation); inhibition of T-cell response, cf. Yanagihava, K.,        Kato, E., Hitomi, S., Sunamoto, J., Wada, H. Glycoconjugate J.        1999, 16, 59-65.    -   Prostaglandin synthesis and COX gene expression is enhanced, cf.        Wu, G., Lu, Z., Ledeen, R. W. Glycoconjugate J. 1996, 13,        235-239.

The biological functions of gangliosides extensively described inliterature are mainly associated with the composition of theoligosaccharide chain. On the other hand, less functional importance isattached to the lipid portion, although it is known that thebio-activity of gangliosides is co-determined by the fatty acidcomposition.

Most previously existing patents therefore deal mainly with theoligosaccharide chain, or do not indicate a specification for the fattyacids, cf. JP 92105616. Others only state the proportion of saturated tounsaturated fatty acids, cf. JP 3101691.

U.S. Pat. No. 5,366,963 22/94 describes the correlation between thefatty acid portion and a chain length of <C18 and >C20 and theimmunosuppressive action of a ganglioside mixture, the fatty acids C18and C20 being explicitly excluded.

It has now surprisingly been found that the lipid component ingangliosides has a much greater importance than was previously assumed.

It is therefore the object of the present invention to provide aganglioside mixture with an improved biological activity.

This object is achieved by the teaching of the claims.

The gangliosides in this case correspond to the following generalformula I:(sugar)-OCH₂—CH(—NH—CO—R¹)—CH(OH)—CH═CH—R²  (I)

This general formula I describes in a formulaic way those gangliosideswhich represent the basis for the inventive mixture. We are notconcerned here with the provision of these types of gangliosides assuch, for the inventive mixture may even consist entirely of knowngangliosides. Rather, the invention consists of the fact that, of thegangliosides comprised by the above formula and the residues defined inthe present documents and comprised by this general formula, the group—CO—R1 is made up of certain fatty acids in certain amounts. Generalformula I hence provides the computational and material reference baseto which the data relating to materials and quantities given hereinrefer. It is, for example, possible to obtain inventive mixtures solelyby re-mixing already known gangliosides or ganglioside mixtures. Ofcourse, modifications etc. of the gangliosides may also be carried out,which will be explained in more detail below.

The residue R² in this case signifies a straight, saturated alkylresidue or a straight alkenyl residue, with both of the residuespossessing 10 or more C-atoms, e.g. up to 30 C-atoms. The residue R² canthus have 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29 and 30 C-atoms. Preferably, the number of C-atoms iseven, e.g. 14, 16 and 18. The alkenyl residue may have one, two or threedouble bond(s).

The residue R¹ signifies a straight, saturated alkyl residue with 10 ormore C-atoms, e.g. up to 30 C-atoms. The residue R¹ may thus have 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29 and 30 C-atoms. Preferably, the number of C-atoms is even, e.g. 18,20, 22 and 24. The residue R¹ together with the keto group bound theretorepresents a fatty acid, which is bound to the residues of the moleculein the form of an amide. This residue —CO—R¹ is designated as acyl-fattyacids in the present documents.

An essential aspect of the present invention is that in the inventiveganglioside mixture, the C20:0 fatty acid makes up at least 10 wt.-% ofthe acyl fatty acids (and thus of the group —CO—R¹, see above). Thisinformation thus concerns only one of the nonpolar aliphatic chains ofthe gangliosides. This means that there is no information given as tothe other nonpolar aliphatic chain and the sugar unit. This otheraliphatic chain may be of a known and/or any desired nature.

C20:0 designates, according to the usual nomenclature, an acyl groupwith 20 C-atoms (counting the carbon atom of the CO-group) withoutdouble bond (:0).

According to a preferred embodiment, the C20:0 fatty acid amounts to 10to 15 wt.-%. With this range indication, all intermediate values, allnarrower ranges, and in particular all integer intermediate values arecomprised and disclosed, e.g. 11, 12, 13 and 14 wt.-%.

According to a preferred embodiment, the weight ratio of the C18:0 acylfatty acid in the —CO—R¹-group to the C20:0 acyl fatty acid in the—CO—R¹-group is 1.0 to 3.0. Also this range indication comprises allintermediate values, all narrower ranges, and in particular singlevalues such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8 and 2.9.

According to a further aspect of the invention, the ganglioside mixtureis characterized in that the group —CO—R¹ may represent a C23:0 fattyacid, which makes up 10 wt.-% or less of the acyl fatty acids in thelipid portion of the ganglioside molecule.

The inventive ganglioside mixture may be animal-based (the term ‘animal’used herein does not denote a human being nor a source of human origin)and/or vegetable-based lipid extracts obtained solely from fungi andmonocellular organisms and/or synthetically produced gangliosides. Inorder to achieve the inventive composition of the acyl fatty acids, thegangliosides from the lipid extracts given above may be modified. Inaddition, it is possible to add isolates of gangliosides with anenriched fatty acid specification to such lipid extracts. It is alsopossible to add chemically synthesized gangliosides to such lipidextracts. Furthermore, the gangliosides making up the mixture may beprepared completely synthetically. It is also possible to combinedifferent types of modifications and/or preparations. Preferably,however, it is assumed that native gangliosides are used. These may thenbe modified. Preferably, the inventive mixtures are thus made fromnative unmodified gangliosides which had been mixed with gangliosidesobtained from native gangliosides and then modified. Some of themodification possibilities are given below:

-   -   Acyl chain specification of native/isolated        gangliosides/ceramides by enzymatically cleaving off the N-acyl        residue (sphingolipid/ceramide N-deacylase EC 351-) and new        specific fatty acid esterification by acid/alkaline catalyzed        chemical esterification or specific fatty acid derivatization        according to Anand, J. K., Sadozai, K. K., Hakomori, S., Lipids        1996, 31, 995-998, by means of free fatty acids.    -   Interesterification of gangliosides by ceramide N-deacylase        EC351 with specific free fatty acids, methyl esters, ethyl        esters or triaglycerides at an optimized pH value and        temperature.    -   Acyl chain specification of native, isolated        gangliosides/ceramides by deacylation to lysogangliosides        according to Gasa, S., Kamio, K., Makita, A. J., Lipid Res.        1992, 33, 1079-1084, followed by an acid/alkaline catalyzed        chemical esterification of specific fatty acids by means of free        fatty acids, methyl esters, ethyl esters or triaglycerides.

These modification possibilities will be discussed in more detail in theexamples below.

By modifying the acyl fatty acids in terms of the invention, thebiological activity of the gangliosides may be enhanced. Thisenhancement of the biological activity applies to the most diverseganglioside functions. If a ganglioside has, for example, a certainbiological activity, by changing or modifying the lipid portion, withoutchanging, for example, the sugar unit, the corresponding, alreadyexisting biological activity will be enhanced. This is based on the factthat due to the inventive modification of the ganglioside lipid portion,an improved mobility of the molecule within the membranes is achieved.The high mobility and the spatial arrangement of gangliosides within themembrane (clustering, cholesterol/glycolipids micro-domains) induced bythe inventive fatty acid composition lead to an enhancement of thereceptor-mediated signals (influences cell surface receptors), theactivity of ion channels (e.g., Na/K ATPase) and the activity ofmembrane-inherent enzymes (e.g. phosphokinase C), cf. Zeller, C. B.,Marchase, R. B., Am. J. Physiol. 1992, 262, C1341-C1355.

Due to the modification of the lipid portion (or more precisely, of theacyl fatty acids), the already existing biological activity ofgangliosides may be enhanced.

The inventive ganglioside mixture may be used in pure form, as well asin the form of an emulsion, in particular an aqueous emulsion. Theganglioside mixture may also be used together with other lipids, itbeing assumed that the further lipids do not influence the biologicalactivity of the ganglioside mixture or of the gangliosides constitutingthis mixture. This means that the biological activity of thegangliosides is only influenced by the fatty acids present in the lipidcomponent of the ganglioside but not by the fatty acids used inaddition.

The inventive ganglioside mixture may be added to any usual foodstuff,and also to a pharmaceutical, if appropriate. In other words, thesubject-matter of the invention also includes food, dietetic andpharmaceutical compositions containing an inventive ganglioside mixture.

These compositions include in particular instant food, food supplementsand formulation food. In this context, a formula food is understood tomean food that has been formulated on the basis of animal (but nothuman) and vegetable starting materials or products. The inventiveganglioside mixture may, for example, be added as admixtures oradditives to the following products, although this enumeration is notlimited: milk and milk products, infant formulae and babyfood, chocolatebars, yoghurt drinks, restorative food, probe food, infusions andproducts for pregnant women.

The inventive ganglioside mixture may also be administered in the formof a pharmaceutical composition alone or together with one or severaladditional active agent(s). These may, for example, be formulated as atablet/capsule. For the formulation of such pharmaceuticals, usualadjuvants, carriers, auxiliary agents, diluents, moisturizing agents,thickening agents, flavoring agents, sweetening agents, etc. may beused.

The pharmaceutical compositions may be administered in any usual way(e.g. parenterally or enterally) to a patient (i.e. human and animal).However, for the sake of convenience, they will be compositions suitedfor oral or lingual administration and formulated to suit the kind ofadministration.

In order to find out whether a mixture containing gangliosides or acomposition containing gangliosides of the kind described here containsan inventive ganglioside mixture, it is only necessary to determine theamount of ganglioside belonging to general formula I, and then toanalyze whether the specifications given here with regard to the acylgroup —CO—R¹ are met.

If compounds or gangliosides are present that do not fall under generalformula I, what is altogether possible, then these are not taken intoaccount in the calculation.

The foods, dietetic compositions and pharmaceutical compositionscontaining the inventive ganglioside mixture, may be used, among otherthings, for improving the development of the gastrointestinal tract, theintestinal immunosystem and the neuronal system, and the treatment ofdestructive changes of the intestinal tract, the intestinal immunosystemand the neuronal system. All age groups, ranging from new born babies upto elderly people, may be mentioned as target groups for the inventiveganglioside mixture.

The invention will be explained in more detail below by means ofexamples, examples 1 to 4 describing methods of producing or modifyingthe inventive gangliosides or ganglioside mixtures, whereas the examples5 to 8 explain application examples or formulation examples for theinventive compositions. The gangliosides of the inventive gangliosidemixture are known compounds or may be produced according to knownmethods.

EXAMPLE 1 Extraction and Separation Starting from Raw Materials

Glycosphingolipids are extracted from natural matrices (egg, soybean,animal milk, colostrum, buttermilk, animal tissues, etc.) according toLadisch and Gillard (Ladisch S., Gillard, B., A solvent partition methodfor morcoscale ganglioside purification, 1984, Analytical Biochemistry146, 220-231) with chloroform-methanol, and are purified by partitioningwith diisopropyl ether-1-butanol aqueous NaCl-solution. Separation ofthe individual ganglioside species having a different saccharidecomposition may be performed using a normal phase HPLC according toGazzotti (Gazzotti, G., Sonnino, S., Ghidoni, R., Normal phase highperformance liquid chromatographic separation of non-derivatizedganglioside mixtures, 1985, J. Chromatogr. 348, 371-78). Separation andfractionation of gangliosides with regard to the acyl chain specificityis carried out by RP-HPLC as follows: gangliosides dissolved in waterare fractionated by means of an RP-8 column with a solvent system ofacetonitrile with 5 mM of a sodium phosphate buffer, pH 7.0. Thechromatogram is recorded by UV/VIS detection (wavelength: 195 nm)according to Wagener (Wagener, R., Kobbe, B., Stoffel, W.,Quantification of gangliosides by microbore high performance liquidchromatography, 1996, J. Lipid Res. 37, 1823-1829) or by a lightscattering detection according to Caboni, (Caboni, M. F., Menofta, S.,Lercker, G., Separation of phospholipids in different foods withlight-scattering detector, 1996, JAOCS 73, 1561-1566). The obtainedfractions are quantified by a five-point calibration of thechromatogram. The individual acyl chain specificities are accumulated bymeans of an automatic fraction collector, and may be recomposedaccording to a mixing ratio of the acyl chain specification.

EXAMPLE 2 Enzymatic Transesterification

Glycosphingolipids are subjected to a transesterification with specificfree fatty acids either directly in the natural matrix (egg, soybean,animal milk, colostrum, buttermilk, animal tissue, etc.) or afterextraction according to Ladisch and Gillard (Ladisch S., Gillard, B., Asolvent partition method for morcoscale ganglioside purification, 1984,Analytical Biochemistry 146, 220-231). Transesterification of thesubstrate takes place by means of a substrate-specificceramide-N-deacylase E.C.3.5.1.—(Sigma-Aldrich, Inc., USA) with specificfree fatty acids at optimal pH values in 0.1% Triton X100 (N-acyl chainhydrolysis at a pH of 5.0-6.0; condensation of the lysosphingolipid withfree fatty acids at pH 7.0) (Ito, M., Kurita, T., Kita, K., A novelenzyme that cleaves the N-acyl linkage of ceramides in variousglycosphingolipids as well as sphingomyelines to produce theirlysoforms, 1995, J. Biol. Chem. 270, 24370-24374; Kita, K., Okino, N.,Ito, M., Reverse hydrolysis reaction of a recombinant alkalineceramidase of Pseudomonas aeruginosa, 2000, Biochim. Biophys. Acta 1485(2-3), 111-120). The modified glycpsphingolipids may either be useddirectly in a mixing ratio of the acyl chain specification, or may berecomposed with other components according to a mixing ratio of the acylchain specification.

EXAMPLE 3 Chemical Fatty Acid Modification

Glycosphingolipids are extracted from natural matrices (egg, soybean,animal milk, colostrum, buttermilk, animal tissue, etc.) according toLadisch and Gillard (Ladisch S., Gillard, B., A solvent partition methodfor morcoscale ganglioside purification, 1984, Analytical Biochemistry146, 220-231) and are purified (see above). In order to change the acylchain specification of isolated gangliosides, the extract is convertedaccording to Gasa, S. (Gasa, S., Kamio, K., Makita, A., Improvedpreparation method for Isogangliosides, 1992, J. Lipid Res. 33,1079-1084) by N-trifluoroacetylation of the sphingosine, followed by anN-acetylation and subsequent mild saponification into correspondingdideacylated gangliosides (protective group). These are chemicallydeacylated into lysogangliosides. The cleavage of the acyl chains fromnative gangliosides may also be performed through a direct chemicalhydrolysis according to Gaver and Sweeley (Gaver, R. C., Sweeley, C. C.,Methods for methanolysis of sphingolipids and direct determination oflong chain bases by gas chromatography, 1995, J. Lipid Res. 24,1389-1397) or by an acidic hydrolysis according to Andu (Kadowaki, H.,Bremer, E. G., Evans, J. E., Jungalwala, F. B., McCluer, R H,Acetonitrile-hydrochloric acid hydrolysis of gangliosides for highperformance liquid chromatographic analysis of their long chain bases,J. Lipid Res. 1983, 24, 1389-1397) by means of incubation of thesubstrate with 0.5M HCl in acetonitrile (or methanol) and 4M H₂O at 75°C. for 2 hours. The resulting lysogangliosides are separated from thefree fatty acids, salts and low-molecular contaminants through anionexchange chromatography by means of aminopropyl phase. The new acylchain specificity of the lysogangliosides with specific free fattyacids, methyl esters or ethyl esters is obtained by an acid/alkalinecatalyzed chemical esterification. It is likewise possible tospecifically acylate the resulting glycosphingolipids chemically withspecific fatty acids according to Anand (Anand, J. K., Sadozai K. K.,Hakomori, S., A simple method for the synthesis of ceramides andradiolabeled analogues, 1996, Lipids 31, 995-998). Using diethylphosphoryl cyanide in combination with triethylamine, the sphingolipidamines are coupled with specific fatty acids. In this way it is possibleto obtain specific N-fatty acid sphingolipids, cerebrosides andgangliosides. The reaction is free of racemates. The desired productsare obtained in a yield of 85-90%. The advantage of this method is thatthe specifically esterified gangliosides are present in a highly pureform. The modified glycosphingolipids may be recomposed with othercomponents according to a mixing ratio of the acyl chain specification.

EXAMPLE 4 Chemical Synthesis

Glycosphingolipids are chemically or chemo-enzymatically synthesizedwith specific fatty acids through a total chemical synthesis accordingto Duclos (Duclos, R. I., The total synthesis of ganglioside GM3, 2000,Carbohydr. Res. 328, 489-507). The glycosphingolipids producedsynthetically in a pure form having a defined fatty acid specificity maybe recomposed with other components according to a mixing ratio of theacyl chain specification.

EXAMPLE 5 Preparation of Baby Formulations/Infant Formulae

For providing glycosphingolipids for the preparation of babyformulations, dried buttermilk material is used. The fatty aciddistribution of ganglioside extracts from buttermilk having a fatdistribution of the N-acyl-fatty acids of C18:0 (10-20 wt.-%) and C20:0(0.01-0.5 wt.-%) based on the total amount of the N-acyl-fatty acids isadjusted by mixing in a ratio of 1:6 with a raw material (see below)modified in its fatty acid distribution and having a fat distribution ofthe N-acyl-fatty acids of C18:0 (6-9 wt.-%) and C20:0 (60-90 wt.-%) to anew N-acyl fatty acid distribution of C18:0 of 1-3 wt.-% and C20:0 of10-15 wt.-%. The raw material modified in its fatty acid distribution ofglycosphingolipids, based on 100 g of solid matter content of aformulation food, is prepared as follows: The glycosphingolipids, mainlyGM1, GM3, GD1a, GD1b from buttermilk (Uelzena, Germany) having a fatportion of 10-15%, are extracted and purified according to Ladisch andGillard (Ladisch S., Gillard, B., A solvent partition method formorcoscale ganglioside purification, 1984, Analytical Biochemistry 146,220-231). 10 g of dried buttermilk material (corresponds approximatelyto 1 liter of milk) are mixed with 50 ml of methanol and 50 ml ofchloroform, extracted for 15 minutes, and subsequently pelletized at 4°C. The supernatant is evaporated to about ¼ of its original volume byrotary evaporation or lyophilization.

Subsequently, the protein portions of the solution are precipitated at−20° C. and pelletized. The supernatant is evaporated until dryness. Forpartitioning, the extract is resuspended with 60 ml of diisopropylether/1-butanol (v/v) 60:40, and subsequently added to 30 ml of water. Aphase separation takes place. The aqueous phase contains theglycosphingolipid portion (about 1 g/l) of the raw material. The fattyacid modification is performed as follows: 1 g of glycosphingolipidextract (about 0.83 mmol at MG 1200) is suspended in 10 ml of 20 mMsodium phosphate solution with 0.1% of Triton X100, 100 Uceramide-N-deacylase is added and incubated at 37° C. for 1-2 hours. Thehydrolysis of the N-acyl-fatty acids takes place at pH 5-6.Subsequently, the solution is adjusted to an end concentration of 25 mMsodium phosphate by addition of sodium phosphate. The specific freefatty acid is added in a molar ratio of 1:10 to the glycosphingolipidused (8.3 mmol C20:0). The transesterification takes place at 37° C. atpH 7-8 over 1-2 hours. The modified glycosphingolipids are purifiedthrough a preparative chromatography (RPC18), the conditioning of thephase taking place with chloroform/methanol/0.1M KCl solution (v/v/v)3:98:74. The reaction solution is applied to the column. The column iswashed with chloroform/methanol/0.1M KCl solution (v/v/v) 3:98:74, waterand methanol/water (v/v) 1:1. The ensuing elution of theglycosphingolipids is carried out with ethanol and chloroform/methanol(v/v) 2:1. The eluate obtained is evaporated until dryness. This processyields approximately 0.8 g of glycosphingolipids per liter of buttermilkhaving a fat distribution of the N-acyl-fatty acids of C18:0 (6-9 wt.-%)and C20:0 (60-90 wt.-%) based on the total amount of the N-acyl-fattyacids.

A protein-adapted infant milk formula (Aptamil® from Milupa) containing11.8 g of protein, 56.9 g of carbohydrates, 24.9 g of fat, 2.5 g ofminerals and vitamins and 45 mg of taurine is prepared in the usualmanner in the form of a bead product. The ganglioside mixture describedin a) is added in an amount of 0.2-500 mg/100 mg based on the solidmatter content of the formula food.

EXAMPLE 6 Food Supplements

Ganglioside extracts from bovine colostrum, egg lecithin or buttermilkwere adjusted by modification of the N-acyl-fatty acid distribution orby mixing a raw material with a raw material (see above) modified in itsfatty acid distribution having a fat distribution of the N-acyl-fattyacids to give a fatty acid distribution of 1-3 wt.-% C18:0 and 10-15wt-% C20:0 based on the total amount of the N-acyl-fatty acids.

The described ganglioside mixture is formulated in softgel capsules with10 to 50 mg/capsule.

EXAMPLE 7 A Product for Pregnant Women

An effervescent tablet (final weight 4.15 g) (Neovin® from Milupa) isprepared in a manner known per se by admixing 10 to 50 mg of thedescribed ganglioside mixture. One tablet per day is dissolved in 150 mlwater and swallowed.

EXAMPLE 8 A Product for the Elderly and Debilitated Persons

A balanced pulverized restorative food (Dilsana® from Milupa) containing22.5 g of protein, 7.7 g of fat, 60.8 g of carbohydrates, 5.4 g ofminerals and vitamins is prepared in a manner known per se byincorporating 0.2-500 mg of the described ganglioside mixture based on100 mg of the solid matter content of the formula food. Up to 3×50 g perday of the food are dissolved in 150 ml water and administered.

1. A Dietetic, pharmaceutical or food composition containing a mixtureof gangliosides of the formula I:(sugar)-OCH2-CH(—NH—CO—R1)-CH(OH)—CH═CH—R2  (I) wherein (sugar)represents a sugar residue, the group —CO—R1 represents an acyl fattyacid bound to the residue of the molecule in the form of an amide, R1represents a straight, saturated alkyl residue having at least 10C-atoms, R2 represents a straight, saturated alkyl residue having atleast 10 C-atoms or a straight alkenyl residue having at least 10C-atoms and one, two or three double bond(s), wherein in thegangliosides of formula I present in said mixture, at least 10 wt.-% ofthe group —CO—R1 and thus of the acyl-fatty acids are a C20:0 fattyacid, and the composition is incorporated in an instant food, a foodsupplement or a formulation food.
 2. The composition according to claim1, wherein the C20:0 fatty acid makes up 10 to 15 wt.-% of the group—CO—R1 and thus of the acyl-fatty acids.
 3. The composition according toclaim 1, wherein the group —CO—R1 includes a C18:0 fatty acid and aC20:0 fatty acid, and the weight ratio of said C18:0 fatty acid to theC20:0 fatty acid of the group —CO—R1 in said mixture is 1.0 to 3.0. 4.The composition according to claim 1, wherein the group —CO—R1 includesa C23:0 fatty acid, which makes up 10 wt.-% or less of the acyl fattyacids in the lipid portion of the ganglioside molecules.
 5. Thecomposition according to claim 1, wherein the ganglioside mixture iscomposed of unmodified gangliosides obtained from naturally existingsources of animal and/or vegetable origin.
 6. The composition accordingto claim 1, wherein the ganglioside mixture is composed of modifiedgangliosides obtained from naturally existing sources of animal and/orvegetable origin.
 7. The composition according to claim 1, which isformulated as an aqueous emulsion.
 8. The composition according to claim1, which is a component of a fat blend.
 9. A Dietetic, pharmaceutical orfood composition containing a mixture of gangliosides of the formula I:(sugar)-OCH2-CH(—NH—CO—R1)-CH(OH)—CH═CH—R2  (I) wherein (sugar)represents a sugar residue, the group —CO—R1 represents an acyl fattyacid bound to the residue of the molecule in the form of an amide, R1represents a straight, saturated alkyl residue having at least 10C-atoms, R2 represents a straight, saturated alkyl residue having atleast 10 C-atoms or a straight alkenyl residue having at least 10C-atoms and one, two or three double bond(s), wherein in thegangliosides of formula I present in said mixture, the group —CO—R1includes a C23:0 fatty acid, which makes up 10 wt.-% or less of the acylfatty acids in the lipid portion of the ganglioside molecules; and thegroup —CO—R1 further includes a C20:0 fatty acid, which makes up 10 to15 wt.-% of the acyl fatty acids.
 10. The composition according to claim9, wherein the group —CO—R1 includes a C18:0 fatty acid and a C20:0fatty acid, and the weight ratio of said C18:0 fatty acid to the C20:0fatty acid of the group —CO—R1 in said mixture is 1.0 to 3.0.
 11. Thecomposition according to claim 9, wherein the ganglioside mixture iscomposed of unmodified gangliosides obtained from naturally existingsources of animal and/or vegetable origin.
 12. The composition accordingto claim 9, wherein the ganglioside mixture is composed of modifiedgangliosides obtained from naturally existing sources of animal and/orvegetable origin.
 13. The composition according to claim 9, which isformulated as an aqueous emulsion.
 14. The composition according toclaim 9, which is a component of a fat blend.
 15. The compositionaccording to claim 9, wherein the composition is incorporated in aninstant food, a food supplement or a formulation food.
 16. A Dietetic,pharmaceutical or food composition containing a mixture of gangliosidesof the formula I:(sugar)-OCH2-CH(—NH—CO—R1)-CH(OH)—CH═CH—R2  (I) wherein (sugar)represents a sugar residue, the group —CO—R1 represents an acyl fattyacid bound to the residue of the molecule in the form of an amide, R1represents a straight, saturated alkyl residue having at least 10C-atoms, R2 represents a straight, saturated alkyl residue having atleast 10 C-atoms or a straight alkenyl residue having at least 10C-atoms and one, two or three double bond(s), wherein in thegangliosides of formula I present in said mixture, at least 10 wt.-% ofthe group —CO—R1 and thus of the acyl-fatty acids are a C20:0 fattyacid, the group —CO—R1 includes a C23:0 fatty acid, which makes up 10wt.-% or less of the acyl fatty acids in the lipid portion of theganglioside molecules, and the group —CO—R1 includes a C18:0 fatty acidand a C20:0 fatty acid, and the weight ratio of said C18:0 fatty acid tothe C20:0 fatty acid of the group —CO—R1 in said mixture is 1.0 to 3.0.17. The composition according to claim 16, wherein the C20:0 fatty acidmakes up 10 to 15 wt.-% of the group —CO—R1 and thus of the acyl-fattyacids.
 18. The composition according to claim 16, wherein theganglioside mixture is composed of unmodified gangliosides obtained fromnaturally existing sources of animal and/or vegetable origin.
 19. Thecomposition according to claim 16, wherein the ganglioside mixture iscomposed of modified gangliosides obtained from naturally existingsources of animal and/or vegetable origin.
 20. The composition accordingto claim 16, which is formulated as an aqueous emulsion.
 21. Thecomposition according to claim 16, which is a component of a fat blend.22. The composition according to claim 16, wherein the composition isincorporated in an instant food, a food supplement or a formulationfood.